We have evidence that TRPV4 channels in the nonpigmented ciliary epithelium (NPE) interact with connexin-50 to form a mechanism that responds to mechanical stimuli. Distortion of NPE cells is capable of causing TRPV4 channel activation which in turn causes hemichannel opening at the aqueous humor-facing surface of the NPE. What this means is the ciliary body, which secretes aqueous humor, has a mechanism capable to sensing and responding to distortion caused by an increase of intraocular pressure. Our working hypothesis is that mechanosensitive hemichannel opening and ATP release are steps in an autocrine feedback loop that reduces Na,K-ATPase activity in the NPE. Here, we propose studies on how the hemichannel opening mechanism senses and responds to a mechanical stimulus (Aim 1). We will characterize how the hemichannel mechanism pivots on TRPV4 activation, the role of connexin-50 vs pannexin-1, how TRPV4 channels respond to mechanical stimuli (cell swelling and stretch), and the electrical conductance signal of the hemichannels. Aim 2 studies will examine how hemichannel opening allows ATP to exit the cell then activate receptors and signaling pathways that change Na,K-ATPase activity in an autocrine fashion. We also will study cAMP and melatonin release into the aqueous humor via hemichannels. Studies in Aim 3 will determine the effect of intraocular pressure on the NPE hemichannel mechanism in an ex vivo arterially perfused eye preparation and test whether the effect of hemichannel blocking molecules and TRPV4-interacting drugs on the rate of aqueous humor formation. The concept of mechanosensitive feedback regulation of Na,K-ATPase activity in the NPE is significant because Na,K-ATPase activity provides the driving force for aqueous humor secretion. The NPE forms a cellular barrier between blood and aqueous and so is subjected to altered physical forces when intraocular pressure changes in relation to hydrostatic pressure in the ciliary process stroma.